The insufficiency of ATG4A in macroautophagy

Nguyen, Nathan; Olivas, Taryn J.; Mires, Antonio; Jin, Jiaxin; Yu, Shenliang; Luan, Lin; Nag, Shanta; Kauffman, Karlina J.; Melia, Thomas J.

doi:10.1074/jbc.ra120.013897

Cited by 10 publications

(6 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This technique involves several centrifugation steps to separate membrane compartments from one another (Figure 3A). Consistent with Strømhaug et al and our own previous results (Jeong et al, 2009;Nguyen et al, 2020;Strømhaug et al, 1998), GFP-LC3B-II and LC3B-II were strongly enriched in fraction 6 in WT cells (Figure 3, B, D, and G), hereafter referred to as the autophagosome (AP) fraction. Strikingly, ATG9A was also robustly enriched in this fraction (Figure 3, B, D and E).…”

Section: Atg9a and Lc3b Reside In The Same Membrane Fraction After De...supporting

confidence: 92%

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes

Olivas

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

During autophagosome biogenesis, the incorporation of transmembrane proteins into the expanding phagophore is not readily observed. In addition, the membrane surface area of the organelle expands rapidly, while the volume of the autophagosome is kept low. Several recent studies have suggested a model of membrane expansion that explains how these attributes are maintained. The autophagosome expands predominantly through the direct protein-mediated transfer of lipids through the lipid transfer protein ATG2. As these lipids are only introduced into the cytoplasmic-facing leaflet of the expanding phagophore, full membrane growth also requires lipid scramblase activity. ATG9 has been demonstrated to harbor scramblase activity and is essential to autophagosome formation, however if and when it is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already fully competent to collect proteins normally found on mature autophagosomes, including LC3-II. Further, through the novel use of styrene-maleic acid lipid particles as a nanoscale interrogation of protein organization on intact membranes, we show that ATG9 is fully integrated in the same membranes as LC3-II, even on maturing autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the rapidly expanding autophagosome membrane. Thus, ATG9 vesicles are the seed membrane from which mammalian autophagosomes form.

show abstract

Section: Atg9a and Lc3b Reside In The Same Membrane Fraction After De...supporting

confidence: 92%

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes

Olivas

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Furthermore, we confirmed that the isoform ATG4B is the major cysteine protease priming the LC3B biosensor, and that its knockout results in a complete lack of priming. Additionally, we provided evidence that ATG4A could mildly contribute to the priming of the LC3B biosensor in the absence of ATG4B, corroborating previous findings concerning a functional redundancy among these isoforms [24, 60]. Interestingly, our biosensor provided novel information on the relevance of specific ATG4B residues for its priming activity.…”

Section: Discussionsupporting

confidence: 88%

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells

Gökerküçük

Chéron

Bertolin

2022

Preprint

View full text Add to dashboard Cite

Although several mechanisms of autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to autophagy activation, the ATG4B protease primes the key autophagy player LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster's Resonance Energy Transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine/tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image allows to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are small pools of unprimed LC3B upon ATG4B downregulation, and that the priming of the biosensor is completely abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Last, we screened for commercially-available ATG4B inhibitors, and we illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.

show abstract

“…While in yeast there is just one ATG8 protein, in mammals there are multiple ATG8 orthologues: LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2 [17]. The ATG8 proteins are synthesised as a precursor and the C‐terminal glycine residue is cleaved by ATG4 to produce the mature ATG8‐I, which will be conjugated to PE, forming the lipidated version ATG8‐II on the membranes of autophagosomal structures [18]. The ATG8 acts as a scaffold for the early core autophagy components by binding protein subunits containing the sequence motif called LC3‐interacting region (LIR) [19].…”

Section: Molecular Mechanisms Of the Autophagic Processmentioning

confidence: 99%

Autophagy at the interface of endothelial cell homeostasis and vascular disease

Mameli

Martello²,

Caporali

2021

The FEBS Journal

View full text Add to dashboard Cite

Autophagy is an essential intracellular process for cellular quality control. It enables cell homeostasis through the selective degradation of harmful protein aggregates and damaged organelles. Autophagy is essential for recycling nutrients, generating energy to maintain cell viability in most tissues and during adverse conditions such as hypoxia/ischaemia. The progressive understanding of the mechanisms modulating autophagy in the vasculature has recently led numerous studies to link intact autophagic responses with endothelial cell (EC) homeostasis and function. Preserved autophagic flux within the ECs has an essential role in maintaining their physiological characteristics, whereas defective autophagy can promote endothelial pro‐inflammatory and atherogenic phenotype. However, we still lack a good knowledge of the complete molecular repertoire controlling various aspects of endothelial autophagy and how this is associated with vascular diseases. Here, we provide an overview of the current state of the art of autophagy in ECs. We review the discoveries that have so far defined autophagy as an essential mechanism in vascular biology and analyse how autophagy influences ECs behaviour in vascular disease. Finally, we emphasise opportunities for compounds to regulate autophagy in ECs and discuss the challenges of exploiting them to resolve vascular disease.

show abstract

The insufficiency of ATG4A in macroautophagy

Cited by 10 publications

References 62 publications

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes

ATG9 vesicles comprise the seed membrane of mammalian autophagosomes

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells

Autophagy at the interface of endothelial cell homeostasis and vascular disease

Contact Info

Product

Resources

About